Favored sites for HIV cDNA integration in the human genome. Abstract Replication of HIV requires integration of a DNA copy of the viral RNA genome into a host cell chromosome. The basic chemical steps mediating action of the viral integrase (IN) protein are now well understood, so interest turns to understanding the roles of cellular factors in the integration process in vivo, and use of such information to develop inhibitors. Raltegravir, the first FDA-approved integrase (IN) inhibitor, binds the enzyme active site and blocks DNA strand transfer. Evolution of HIV in the presence of raltegravir elicits resistance mutants, however, motivating studies of potential new drug targets in the preintegration complex. IN binds the host cell protein PSIP1/LEDGF/p75 (henceforth LEDGF), which is important for efficient integration, and previous work under this award showed that LEDGF targets HIV integration to active transcription units via a tethering mechanism. We also reported that the LEDGF binding site on IN can be bound by small molecule inhibitors, and now highly active inhibitors targeting this site are available. This application thus focuses on identifying interactions between HIV IN and host factors important in integration (Aim 1), and studies of integrase inhibitors (Aim 2) with the dual goals of optimizing therapy and using inhibitors to understand mechanism. Research in both aims will be driven in part by our infrastructure for studying retroviral integration targeting, which consists of well established bioinformatic pipelines and an archive of over one billion integration site sequence reads. Progress from the previous funding cycle allows us to propose several exciting new hypotheses to investigate in the renewal. 1) We hypothesize that HIV PICs engage in an orderly series of interactions with cellular proteins, beginning in the cytoplasm, that ultimately delivers PICs to favored integration sites in the nucleus. 2) We hypothesize that an inhibitor that binds to the LEDGF interacting site on integrase can have wider effects on the HIV replication cycle than previously appreciated, sending us in new directions in the study of LEDGF. 3) We hypothesize that raltegravir alters integration targeting by increasing the proportion of events mediated by the cellular NHEJ machinery. Thus our Specific Aims are: Aim 1. Mechanistic studies of interactions between PICs and cellular factors to identify potential inhibitor targets. Aim 2. Probing the mechanism of integrase inhibitors, both those targeting the LEDGF binding site and the active site. !